SNP (Single Nucleotide Polymorphism) molecular marker related to birth weight of pig and application thereof

Abstract

The invention belongs to the technical field of molecular biotechnology and molecular markers, and discloses a pig birth weight related SNP (single nucleotide polymorphism) molecular marker and application thereof, the pig birth weight related SNP molecular marker corresponds to a nucleic acid site at 5379864 bp of chromosome 16 of a pig reference genome Sscrofa11.1 version sequence, and a basic group of the site is C or T and is contained in an intron of an MARCHF11 gene. The invention also discloses an application of the SNP molecular marker related to the birth weight of the pig in genetic breeding of landrace pigs, and provides a method for breeding or assisted breeding of pigs with high / low birth weight by using the SNP molecular marker and a primer for identifying the SNP site. By means of the method, precise prediction and efficient selection of the birth weight of the landrace can be achieved, breeding of excellent breeding pigs is promoted, the population improvement process is accelerated, and powerful support is provided for sustainable development of the pig breeding industry.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology and molecular marker technology, and in particular to a SNP molecular marker associated with pig birth weight and an application thereof. Background Art

[0002] Landrace pig is a widely used lean pig breed. It occupies an important position in the global pig industry with its good growth performance, high lean meat rate and adaptability. The birth weight of pigs is an important trait that affects individual growth and development, production performance and health status, and is directly related to the economic benefits of breeding. Low birth weight will lead to slow growth of piglets in the early stage, frequent health problems, and low feed conversion rate, which will eventually affect the market weight and slaughter rate. Therefore, molecular marker-assisted breeding based on birth weight is of great significance in improving the birth weight of Landrace pig populations and optimizing production performance.

[0003] However, no effective SNP molecular markers related to birth weight of Landrace pigs have been found so far, so the existing technology needs to be further improved. Summary of the invention

[0004] In response to the above problems, the present invention provides a SNP molecular marker related to pig birth weight and its application. The SNP molecular marker related to pig birth weight can be applied to the selection or auxiliary selection of pigs with high / low birth weight, and can be used for the early selection of high-quality pig breeding, shortening the breeding cycle, and accelerating the population improvement process.

[0005] To solve the above problems, this application provides the following technical solutions: In the first aspect, the present application provides a SNP molecular marker associated with pig birth weight, the SNP site corresponds to the nucleic acid site at 5379864 bp on chromosome 16 of the pig reference genome Sscrofa11.1 version sequence, the base of the site is C or T, and is contained in the intron of the MARCHF11 gene.

[0006] As a member of the MARCH family, the MARCHF11 (Membrane Associated Ring-CH-Type Finger 11) gene plays multiple roles in cell growth, differentiation, apoptosis and immune regulation. MARCHF11 encodes a protein with E3 ubiquitin ligase activity, which can attach ubiquitin tags to specific target proteins, thereby regulating the degradation, localization and signal transduction process of the target protein. The ubiquitination process plays an indispensable role in key biological processes such as early embryonic cell proliferation and differentiation, and may play a potential role in birth weight and embryonic development, which is also consistent with the above-mentioned SNP sites related to pig birth weight in the MARCHF11 gene screened in this application.

[0007] In marker-assisted selection (MAS), molecular markers improve breeding accuracy and efficiency by identifying genetic variants that are closely associated with target traits. Molecular markers based on the MARCHF11 gene can assist in the screening of Landrace pig populations with higher birth weights, and optimize the birth weight and growth performance potential of breeding pigs through rapid evaluation of individual genotypes. The application of this molecular marker can significantly shorten the breeding cycle, increase the rate of genetic improvement, and ultimately bring higher economic benefits and a better supply of pork products to the breeding industry. Therefore, the MARCHF11 gene has a high application potential in the birth weight of Landrace pigs, and its development and application as a molecular marker is expected to provide a new approach for Landrace pig breeding.

[0008] This application is the first to identify a SNP (g. 5379864 C>T) site in the MARCHF11 gene that is significantly associated with the birth weight of Landrace pigs through a whole genome association analysis method. This site can be applied to the genetic improvement of growth and reproduction traits of Landrace pigs, providing a new genetic marker resource for molecular marker-assisted selection of Landrace birth weight. The identified SNP site associated with birth weight can be used as a molecular marker for the selection of excellent traits of Landrace pigs, which will help accelerate the genetic improvement of production performance of Landrace pigs.

[0009] In a second aspect, the present application also provides the application of the above-mentioned SNP molecular markers related to pig birth weight in pig genetic breeding.

[0010] Specifically, the upstream and downstream nucleotide sequences of the SNP molecular marker related to pig birth weight are shown in SEQ ID NO: 1, the SNP site is the 32nd base of the sequence, and the single nucleotide at this site is a mutation of T base to C, which leads to differences in the birth weight of Landrace pigs.

[0011] The aforementioned SNP molecular markers associated with pig birth weight can be used for the selection or auxiliary selection of breeding pigs with high / low birth weight, which will help accelerate the progress of genetic improvement of the production performance of Landrace pigs.

[0012] In a third aspect, the present application also provides a method for breeding or assisting in breeding pigs with high / low birth weight using the above-mentioned SNP molecular markers related to pig birth weight, which comprises the following steps: The aforementioned pig birth weight-related SNP molecular marker at 5379864 bp on chromosome 16 of the pig genome is detected. The base of the site is C or T, so as to determine whether the genotype of the pig to be tested is CC, CT or TT, and then select pigs with TT genotype or CC genotype for the next step of breeding and / or breeding according to production needs.

[0013] Optionally, in the method of using the aforementioned SNP molecular markers associated with pig birth weight to select or assist in the selection of pigs with high / low birth weight, if breeding pigs with high birth weight, select Landrace pigs with CC genotype; if breeding pigs with low birth weight, select pigs with TT genotype.

[0014] The birth weight of the TT genotype Landrace pig is higher than that of the CT genotype Landrace pig, and the birth weight of the CT genotype Landrace pig is higher than that of the CC genotype Landrace pig.

[0015] Optionally, in the application of the above SNP molecular marker in pig genetic breeding or in the method of using the above SNP molecular marker in breeding or assisting breeding of pigs with high / low birth weight, the pig is a Landrace pig.

[0016] In a fourth aspect, the present application also provides a primer pair for the aforementioned pig birth weight-related SNP molecular marker, the primer pair comprising: an upstream primer F1 with a sequence as shown in SEQ ID NO: 2 and a downstream primer R1 with a sequence as shown in SEQ ID NO: 3.

[0017] In a fifth aspect, the present application also provides the use of the above primer pair in the selection or auxiliary selection of pigs with high / low birth weight.

[0018] In a sixth aspect, the present application also provides a kit for detecting the above-mentioned SNP molecular markers, wherein the kit contains the above-mentioned primer pair.

[0019] In a seventh aspect, the present application further provides an application of the above-mentioned SNP molecular marker in breeding high birth weight pig strains, the application comprising the following steps: (1) Detecting the aforementioned SNP molecular markers related to pig birth weight in reserve breeding pigs; (2) Selecting and retaining individuals with the TT genotype detected in step (1) as breeding pigs, and mating the breeding boars and sows; (3) The aforementioned SNP molecular markers are detected in the piglets born from the mating in step (2), and the individuals with the genotype of the locus are retained, and the individuals with the genotype of CC are eliminated, so as to breed a pig strain with high birth weight.

[0020] Optionally, the application further comprises: repeating the operations of steps (2) to (3) with the individuals bred in step (3), thereby selecting breeding pig individuals with a molecular marker genotype of TT in the offspring and eliminating individuals with a CC genotype, so as to increase the frequency of the allele T in the offspring pig population generation by generation and increase the birth weight of the offspring pigs.

[0021] In an eighth aspect, the present application also provides a method for screening SNP molecular markers associated with pig birth weight, comprising the following steps: (1) Record and count the birth weight information of each individual in the pig test group, including various influencing factors (such as field, year, season, parity, sex, etc.), and use the least square mean (LSM) hypothesis test and multiple comparison method to detect whether the influencing factors reach a significant level (P ≤ 0.05); (2) Collect pig tissue or blood samples, extract genomic DNA and test its quality, and use the pig "SMIC-1" 50K chip for genotyping; then perform quality control on the genotyping data, filter out SNP sites that do not meet the requirements, and then exclude individuals with a sample detection rate of less than 95% from the sample; (3) Fixed and random model circulating probability unification (FarmCPU) was used to perform genome-wide association analysis to screen SNPs, key candidate genes and molecular markers associated with pig birth weight.

[0022] Optionally, in the method for screening SNP molecular markers related to pig birth weight, the SNP sites that do not meet the requirements are: SNP sites with a call rate (CR) less than 95%, a minimum gene frequency (MAF) less than 0.05 and no map information.

[0023] Preferably, the pig is a Landrace pig.

[0024] The present invention has the following beneficial effects: 1. The present invention discovered for the first time a site in which the MARCHF11 gene is associated with the birth weight of Landrace pigs, providing an effective candidate gene for improving the excellent growth and reproduction traits of Landrace pigs. At the same time, the present invention used a whole genome association analysis method to identify a SNP (g.5379864 T>C) in the MARCHF11 gene that was significantly associated with the birth weight of Landrace pigs. It was found that the birth weight of Landrace pigs with the TT genotype at this site was higher than that of Landrace pigs with the CT genotype, and the birth weight of Landrace pigs with the CT genotype was higher than that of Landrace pigs with the CC genotype. Therefore, this site can be applied to the genetic improvement of the growth and reproduction traits of Landrace pigs, providing a new genetic marker resource for molecular marker-assisted selection of Landrace birth weight.

[0025] 2. Based on the discovery of the SNP molecular markers associated with the birth weight of Landrace pigs, the present application also provides a method for using the SNP molecular markers associated with the birth weight of Landrace pigs to select or assist in the selection of Landrace pigs with high / low birth weights, which helps to accelerate the genetic improvement of the production performance of Landrace pigs. In order to achieve accurate prediction and efficient selection of Landrace pig birth weight, early selection of high-quality breeding pigs, promote the cultivation of excellent breeding pigs, shorten the breeding cycle, and provide strong support for the sustainable development of the pig farming industry.

[0026] 3. The invention also provides a method for screening candidate markers and genes related to birth weight of Landrace pigs based on a method of whole genome association analysis. The SNP sites related to birth weight identified by screening can be used as molecular markers for the selection of excellent traits of Landrace pigs, which will help accelerate the genetic improvement of production performance of Landrace pigs. The above screening method has the characteristics of simple operation, comprehensive coverage, and high accuracy of selected markers, and also provides technical support for the genetic analysis of other important economic traits of livestock and poultry. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 To screen out the Manhattan plot of genome-wide association analysis of MARCHF11 genes and SNP loci associated with birth weight in Landrace pigs; Figure 2 This is the QQ plot of the whole genome association analysis of Landrace pig birth weight; Figure 3 The genomic distribution map of SNP loci used in the whole gene association analysis of birth weight in Landrace pigs; Figure 4 This is the principal component analysis (PCA) diagram of the Landrace pig test group; Figure 5 Analysis of birth weight differences between different genotypes of SNP molecular marker g. 5379864 T>C (CNC10160101). DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0029] Example 1 Screening Method for Candidate Genes and SNP Molecular Markers Related to Birth Weight of Landrace Pigs 1. Statistical analysis of the population to be tested and phenotypic data A group of Landrace pigs from a large domestic breeding farm was selected for testing, with a group sample size of 1145 pigs. The birth weight of Landrace pigs was weighed and recorded immediately after birth, and the environmental factors (influencing factors) affecting the birth weight, including the field, year, season, parity, gender and other influencing factors, were recorded at the same time. The phenotypic data were subjected to quality control analysis, and outliers outside the mean value  2 standard deviation were eliminated, and 1134 birth weight records were finally retained. The average birth weight of the Landrace pig group used in this example was 1.42  0.19 kg, with a minimum of 1.00 kg and a maximum of 2.20 kg.

[0030] 2. Statistical analysis of factors affecting birth weight The least square mean (LSM) hypothesis test and multiple comparison analysis (Tukey's HSD) were used to evaluate the significant influence level of various influencing factors on birth weight. The R package emmeans was used for fitting correction, and the linear model and subsequent genome-wide association analysis model were included only when and only when the factors affecting each trait reached a significant level (P ≤ 0.05). In this embodiment, the environmental factors that have a specific significant effect on the birth weight of Landrace pigs after analysis include field, year, season, parity, and sex (as shown in Table 1).

[0031] Table 1 Results of least squares means multiple comparisons of factors affecting birth weight of Landrace pigs Traits / Factors Sessions years season Parity gender Birth weight (Kg) 0.0037** 0.0001*** 7.23E-05*** 0.0015** 0.0001*** Note: *** means P ≤ 0.001; ** means 0.001 ≤ P ≤ 0.01; * means 0.01≤P≤0.05.

[0032] 3. Sample Collection and DNA Extraction Landrace pig ear tissue samples were collected and immediately stored at -80°C until subsequent DNA extraction. Genomic DNA was extracted using a genomic DNA tissue extraction kit in strict accordance with the instructions of the reagent. DNA purity was determined using a NanoDrop 2000 UV spectrophotometer to ensure that the OD260 / OD280 ratio was between 1.8 and 2.0, and DNA integrity was detected by 1% agarose gel electrophoresis to ensure that the DNA quality met the requirements for subsequent chip analysis.

[0033] 4. Genotyping and quality control The "SMIC-1" pig 50K SNP chip was used for genotyping, covering a total of about 56,000 SNP sites in the whole genome for subsequent genome-wide association analysis (GWAS). Through steps such as DNA amplification, fragmentation, resuspension, chip hybridization, chip cleaning, single base extension, and chip scanning, the results were imported into the signal recognition software for visualization processing, and finally the genotyping data of each sample was obtained.

[0034] PLINK v1.9 was used to perform quality control on the genotype data, and sites with SNP site detection rates below 95%, SNP sites with minimum gene frequencies below 0.01, and SNP sites without map information were removed. Quality control was also performed on individual samples, and individuals with genotype detection rates below 95% were removed. Finally, a total of 46,829 high-quality SNP sites and 1,145 samples passed quality control (such as Figure 3 as shown).

[0035] 5. Genome-wide association analysis This example uses the fixed and random model cyclic probability unified method (FarmCPU) to analyze the population principal component analysis results (PCA=3, such as Figure 4 As shown in the figure, the significant influencing factors of birth weight, including field, year, season, parity, and sex, were added as fixed effects into the whole genome association analysis model to screen the SNP sites significantly associated with the birth weight of Landrace pigs (such as Figure 1 , 2 Bonferroni multiple test correction was performed on the candidate SNPs, and P < 0.05 / effective SNP number was set as the significance threshold to ensure the reliability of the analysis results.

[0036] After testing, a total of 6 SNPs were obtained that were significantly associated with the birth weight of Landrace pigs (as shown in Table 2). Based on the pig reference genome (Sscrofa11.1 version) information, the MARCHF11 gene was further located as a candidate gene. Among them, CNC10160101 is a candidate marker associated with birth weight. The marker is located at 5379864 bp on chromosome 16, and mutates from cytosine C to thymine T, expressed as g. 5379864 T>C.

[0037] Table 2 SNP markers and candidate genes associated with birth weight in Landrace pigs Marker site chromosome Site information (bp) Allele P-value Genes within the locus CNC10031242 3 61739022 A / G 1.29E-07 -- CNC10032309 3 117474270 T / G 7.51E-07 -- CNC10060592 6 28761105 T / A 2.44E-09 -- CNC10082703 8 133752515 C / T 1.60E-07 -- CNC10160101 16 5379864 T / C 7.56E-07 MARCHF11 6. Molecular marker application and verification The candidate marker site g. 5379864 T>C was screened out and the corresponding genotypes (CC, CT, TT) were subjected to variance analysis (ANOVA) with the birth weight of Landrace pigs. The Tukey HSD test was further used to compare the significance of the differences between the different genotypes (as shown in Table 3). The R package ggplot2 was used to draw the violin plot and add significant markers (such as Figure 5 as shown).

[0038] Table 3 Analysis of birth weight differences among different genotypes of Landrace pigs with molecular marker g. 5379864 T>C genotype Number of individuals Genotype frequency (%) Birth weight (Kg) Multiple comparison results TT 473 41.31 1.390.17 TT vs. CC: ** CT 538 46.99 1.420.21 CT vs. CC: ** CC 134 11.70 1.480.20 CT vs. CC: * Note: ** indicates that the birth weights of different genotypes are extremely significantly different (P ≤ 0.01); * indicates that the birth weights of different genotypes are significantly different (P ≤ 0.05).

[0039] The analysis results showed that at the marker site g. 5379864 T>C, the birth weight of Landrace pigs with TT genotype was 1.480.20 Kg, which was significantly higher than that of CT genotype (1.420.21 Kg), and extremely significantly higher than that of CC genotype (1.390.17 Kg). Therefore, the molecular marker g. 5379864 T>C can be used for molecular marker-assisted selection of Landrace pig birth weight, and individuals with TT genotype can be selected for the next step of selection or breeding to improve the birth weight trait of Landrace pigs.

[0040] Example 2 Application of SNP molecular markers associated with birth weight of Landrace pigs in breeding Landrace pigs with high / low birth weight This example is the design of upstream and downstream primers and amplification results for the detection of C / T at the SNP site 5379864 bp on chromosome 16 obtained in Example 1 1. Primer design of target gene sequence According to the target gene sequence, the optimized amplification primers were designed using primer design software. The primer pair sequences are as follows: Upstream primer: 5'-ACAGTCACCTTGGGGTTTCC-3' (as shown in SEQ ID NO: 2) Downstream primer: 5'-AGAGGTTTCCCTTCCCCCTT-3' (as shown in SEQ ID NO: 3) 2. Landrace Pig Genome Extraction Collect ear tissue samples or blood samples from the Landrace pigs to be tested, extract genomic DNA, and store them at -20°C for later use after quality and concentration testing.

[0041] 3. PCR amplification of SNP loci associated with birth weight in Landrace pigs In the experiment, DNA samples of Landrace pig blood genes were mixed to prepare a mixed pool DNA template, and the above primers were used for PCR amplification.

[0042] PCR amplification system (total volume is 20.0 μL): 10.0 μL of 2xTaq PCR Master Mix, 0.5 μL of upstream primer (10 μmol / L), 0.5 μL of downstream primer (10 μmol / L), 1.0 μL of genomic DNA template, 8.0 μL of ddH 2 O.

[0043] The PCR amplification procedure is: Pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, for a total of 30 cycles; extension at 72°C for 5 min.

[0044] The PCR products were detected by 1.5% agarose gel electrophoresis and then subjected to first-generation Sanger sequencing.

[0045] 4. Sequencing and analysis of amplified products The PCR product was detected by agarose gel electrophoresis, and the size of the amplified target fragment was 113 bp (as shown in SEQ ID NO: 6). The amplified product was sent for sequencing, and the sequence of the amplified product was obtained by sequencing. The sequencing result was compared and analyzed with the relevant gene fragments of Landrace pigs in GenBank (TT genotype is shown in SEQ ID NO: 4, and CC genotype sequence is shown in SEQ ID NO: 5) to determine whether the genotype of the target SNP site related to birth weight in the sequence is CC, CT or TT.

[0046] According to the above results, high / low birth weight Landrace pigs were selected. If high birth weight Landrace pigs were selected, Landrace pigs with TT genotype were selected; if low birth weight Landrace pigs were selected, Landrace pigs with CC genotype were selected.

[0047] Example 3 It is easy for a person skilled in the art to refer to other primers designed according to the aforementioned SNP sites of the present invention or probes for identifying the molecular markers, so as to detect the genetic markers, for example, by PCR amplification to obtain the molecular genetic markers, and then by cloning and sequencing to obtain the corresponding sequences, or by RFLP polymorphism detection. Therefore, the present invention also includes other primers for amplifying the molecular genetic markers or probes for identifying the molecular genetic markers, and a kit containing the primers or probes.

[0048] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A SNP molecular marker associated with pig birth weight, characterized in that: Its SNP site corresponds to the nucleic acid site at 5379864 bp on chromosome 16 of the pig reference genome Sscrofa11.1 version sequence. The base of this site is C or T and is contained in the intron of the MARCHF11 gene.

2. Use of the pig birth weight-related SNP molecular marker as described in claim 1 in pig genetic breeding.

3. A method for breeding or assisting in breeding pigs with high / low birth weight using the pig birth weight-related SNP molecular markers as claimed in claim 1, characterized in that: The following steps are involved: The SNP molecular marker associated with pig birth weight as described in claim 1 is detected at 5379864 bp on chromosome 16 of the pig genome, and the base of the site is C or T, thereby determining whether the genotype of the Landrace pig to be tested is CC, CT or TT, and then selecting Landrace pigs with TT genotype or CC genotype for the next step of seed selection and / or breeding according to production needs.

4. The method according to claim 3, characterized in that If you want to breed pigs with high birth weight, choose pigs with TT genotype; if you want to breed pigs with low birth weight, choose pigs with CC genotype.

5. The use of the SNP molecular marker according to claim 1 in pig genetic breeding or the method of the SNP molecular marker according to claim 3 in breeding or assisting breeding of pigs with high / low birth weight, characterized in that: The pig is a Landrace pig.

6. A primer pair for identifying the SNP molecular marker associated with pig birth weight as claimed in claim 1, characterized in that: The primer pair comprises: an upstream primer F1 whose sequence is shown in SEQ ID NO: 2 and a downstream primer R1 whose sequence is shown in SEQ ID NO:

3.

7. A kit for detecting the SNP molecular marker according to claim 1, wherein: The kit contains the primer pair as claimed in claim 5.

8. Use of the primer pair as claimed in claim 6 in breeding or assisting in breeding of pigs with high / low birth weight.

9. The use of the SNP molecular marker according to claim 1 in breeding high birth weight pig strains, characterized in that: The application comprises the following steps: (1) Detecting the SNP molecular marker associated with pig birth weight as described in claim 1 in reserve breeding pigs; (2) selecting and retaining the individuals with the TT genotype detected in step (1) as breeding pigs, and mating the breeding boars and sows; (3) The SNP molecular marker as described in claim 1 is detected in the piglets born from the mating in step (2), the individuals with the genotype of the site being TT are retained, and the individuals with the genotype being CC are eliminated, so as to breed a pig strain with high birth weight.

10. A method for screening SNP molecular markers associated with pig birth weight, characterized in that: The following steps are involved: (1) Record and count the birth weight information of each individual in the pig test group, including various influencing factors, using the least squares mean; hypothesis testing and multiple comparison methods to detect whether the influencing factors reach a significant level; (2) Collect pig tissue samples or blood samples, extract genomic DNA, test its quality, and perform genotyping; then perform quality control on the genotyping data, filter out SNP sites that do not meet the requirements, and exclude individuals with a sample detection rate of less than 95% from the sample; (3) A genome-wide association analysis was performed using a unified method of fixed and random model circular probabilities to screen SNPs, key candidate genes, and molecular markers associated with pig birth weight.

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